Artificial culture of the sexual stage of lagenidium giganteum

ABSTRACT

A method is provided for producing oospores of the organism Lagenidium giganteum comprising the steps of incubating zoospores with a medium containing a sterol and particular fatty acids or fatty acid derivatives.

This invention was made with Government support under subcontract L800200 RF, Project 4148, under Contract OMB 81-2235 USC 200-206 awarded by the Environmental Protection Agency. The Government has certain rights in this invention.

This is a continuation-in-part of Ser. No. 431,176, filed Sept. 30, 1982, now abandoned.

The present invention is directed to a method for producing the sexual stage of the organism Lagendium giganteum. In particular, the present invention is directed to a method of producing oospores in vitro by incubating zoospores or hyphal segments of L. giganteum in a medium containing a particular unsaturated fatty acid, unsaturated fatty acid glyceride or unsaturated fatty acid polyglyceride.

The fungus Lagenidium giganteum is a facultative endoparasite of anopheline and culicine mosquito larvae. The zoospores of L. giganteum actively seek out mosquito hosts and the fungus persists in an active state as long as susceptible hosts are present. If the water source in which the mosquito larvae live dries out, the fungus remains in a dormant state in the form of oospores. Therefore it is desirable to be able to artificially cultivate the oospores of L. giganteum on large scale in order to utilize it as a biological insecticide for mosquito larvae.

The only currently registered biological insecticide for mosquito larvae is the bacterium Bacillus thuringiensis israelensis (BTI). The disadvantage of BTI is that it persists for a maximum of about 48 hours following application under field conditions and it must be ingested by the mosquito larvae.

It is therefore an object of the present invention to provide a method for producing oospores of L. giganteum in vitro whereby oosporogenesis is artificially induced by exogenous components.

The fungus L. giganteum may undergo either an asexual or sexual reproductive cycle. In the asexual cycle motile zoospores of L. giganteum selectively encyst on the cuticle of mosquito larvae, which is pierced enzymatically and mechanically by the fungus. The fungal mycelium subsequently ramifies throughout the larvae until the larval body cavity is completely filled with septate hyphae of the fungus. Hypha segments can develop into zoosporangia and zoospores may be discharged through an exit tube into the environment, thereby continuing the asexual infection cycle. Alternatively, adjacent hyphal segments of the fungus can develop into antheridia (male organs in male sexual cells) and oogonia (female sexual organs in female sexual cells). Subsequent fusion of the protoplasts and nuclei of adjacent hyphal segments by plasmogamy and karyogamy results in thick-walled oospores. The oospores may remain dormant until the correct environmental conditions induce germination, resulting in motile zoospores to initiate the infection cycle.

The present invention is directed to a method for producing oospores of the organism L. giganteum comprising the step of inducing oospore formation by contacting the zoospores with a medium containing a particular unsaturated fatty acid or unsaturated fatty acid derivative, or mixture thereof.

The process for artificial culturing according to the present invention may be conducted on agar media or in liquid culture utilizing rotary shakers or industrial scale fermentation technology. Generally, the process according to the present invention involves growing L giganteum in a growth medium, inducing zoosporogenesis and introducing the resultant zoospores to a nutrient medium to induce oosporogenesis.

According to the present invention, cultures of L. giganteum may be maintained on a modified peptone yeast extract-glucose (PYG) agar medium as disclosed by Brown et al. Amer. J. Bot. J., 42:337-341 (1955), supplemented with 1.5% (w/v) Mazola® corn oil. This commercial corn oil contains a mixture of sterols including stigmasterol, beta-sitosterol, and gamma-sitosterol or campesterol, and C-16 and C-18 saturated and unsaturated fatty acids. Alternatively, the organism may be maintained on an agar medium of the following composition: 1.25 g D-glucose, 1.25 g Difco peptone, 1.25 g Ardamine pH (autolyzed yeast extract, Desmo Chemical Corporation, Elmsford, NY), 2.0 g corn oil, 1.0 g Grumbacher® linseed oil, 0.075 g CaCl₂.2H₂ O and 20 g Difco agar per liter of deionized water.

Zoosporogenesis may be induced by placing a small amount of surface mycelium of hyphal segments from 7- to 14-day old cultures of L. giganteum into sterile deionized water in a 10-cm-diam petri dish. Sufficient densities of zoospores (1.5-2.0×10⁴ /ml) are available for further transfer into the oospore-inducing medium within 12 to 18 hours.

To induce oospore production 8 ml of zoospore suspension (1.2-1.6×10⁵ zoospores) may be inoculated into each 100 ml of oospore-inducing media. Cultures may be incubated for six days on a rotary shaker at 110±5 rpm under ambient conditions of temperature (22°±5° C.) and light. Quantitative oospore yields may be obtained using a hemocytometer beginning a minimum of 10 days post-inoculation. Oospore viability may be determined visually. Cultures may be homogenized, for instance, at high speed for ca. 30 seconds in an Osterizer blender prior to counting.

The oospore-inducing medium according to the present invention comprises an assimilable source of carbon, such as glucose, sucrose, and the like; an assimilable source of nitrogen; a sterol; a non-toxic calcium or magnesium salt; and an unsaturated fatty acid or derivative selected from the group consisting of oleic acid, palmitoleic acid, oleic acid esters, palmitoleic acid esters, linoleic acid esters, diolein, triolein, trilinolein and mixtures thereof. The sterol may be cholesterol or a cholesterol-like sterol, such as, 7-dehydrocholesterol, sitosterol, desmosterol, fucosterol, stigmasterol, cholesteryl acetate, cholesteryl palmitate, and the like. Cholesterol is preferred. The non-toxic salts are preferably the chlorides of calcium or magnesium. The medium may also contain other convention components, such as buffers. A preferred group of fatty acid or derivative compounds comprises palmitoleic acid, diolein, triolein, trilinolein or mixtures thereof.

The following Table 1 illustrates the effect of cholesterol, cholesteryl esters, fatty acids and fatty acid derivatives on in vitro oosporogenesis of L. giganteum.

                                      TABLE 1                                      __________________________________________________________________________     EFFECT OF CHOLESTEROL ESTERIFICATION AND                                       CHOLESTEROL AND FATTY ACID CONCENTATION                                        ON OOSPOROGENESIS                                                                                           Oospore Yield                                     Sterol/Concn. Fatty Acid/    (×10.sup.-6).sup.2                          (mg/L).sup.1  Concn. (g/L)   % Viability                                       __________________________________________________________________________     Sterols and Sterols Esters                                                     Cholesterol/5 --             0                                                 Cholesterol/10                                                                               --             0.05/0                                            Cholesterol/50                                                                               --             0.40/0                                            Cholesterol/100                                                                              --             0.23/0                                            Cholesteryl Oleate/10                                                                        --             0.23/0                                            Cholesteryl Oleate/25                                                                        --             0.18/0                                            Cholesteryl Oleate/50                                                                        --             0.03/0                                            Cholesteryl Oleate/100                                                                       --             0.05/0                                            Cholesteryl Linoleate/10                                                                     --             0.15/0                                            Cholesteryl Linoleate/25                                                                     --             0.61/0                                            Cholesteryl Linoleate/50                                                                     --             0/0                                               Cholesteryl Linoleate/100                                                                    --             0.05/0                                            Sterols and Unsaturated Fatty Acids                                            Cholesterol/25                                                                               Myristic/1.0   0.15/48                                           "             Trimyristion/1.0                                                                              0.40/50                                           "             Palmitic/1.0   0.10/25                                           "             Tripalmitin/1.0                                                                               0.87/16                                           "             Stearic/1.0    0.46/24                                           Cholesterol 25                                                                               Tristearin/1.0 0.12/17                                           "             Arachidic/1.0  0.13/0                                            "             Palmitoleic/0.25                                                                              6.1/6                                             "             Palmitoleic/0.5                                                                               5.7/72                                            "             Tripalmitolein/0.25                                                                           4.2/48                                            "             Oleic/0.1      0.32/5                                            "             Oleic/1.0      1.0/30                                            "             Monoolein/0.25 0.5/0                                             "             Monoolein/0.5  0.83/0                                            -             Monoolein/1.0  3.2/8                                             "             Diolein/0.25   2.1/16                                            "             Diolein/0.5    2.9/62                                            "             Triolein/0.1   3.6/8                                             "             Triolein/0.25  3.1/23                                            "             Triolein/0.5   4.2/72                                            "             Linoleic/0.1   0.33/15                                           "             Linoleic/0.25  0/0                                               "             Linoleic/1.0   TOXIC                                             "             Monolinolein/0.25                                                                             0.49/0                                            "             Monolinolein/1.0                                                                              1.7/0                                             "             Dilinolein/0.25                                                                               0.38/22                                           "             Dilinolein/1.0 1.8/39                                            "             Trilinolein/0.1                                                                               3.8/0                                             "             Trilinolein/0.25                                                                              4.5/37                                            "             Trilinolein/0.5                                                                               6.2/78                                            Cholesterol 25                                                                               Trillinolein/0.1                                                                              4.7/72                                            "             Linolenic/1.0  TOXIC                                             "             Trilinolenin/0.1                                                                              0                                                 "             Trilinolenin/0.25                                                                             0.15/11                                           "             Trilinolenin/0.5                                                                              0.90/3                                            "             Trilinolenin/1.0                                                                              0.90/0                                            "             11,14,17-Eicosatrienoic/0.25                                                                  0/0                                               Sterols, Saturated and Unsaturated Fatty Acids                                 Cholesterol 25                                                                               Triolein/0.25  0.43/0                                                          Tripalmitin/0.1                                                  "             Triolein/0.25  0.33/0                                                          Stearic/0.1    0.33/0                                            "             Triolein/0.25  0.62/27                                                         Tristearin/0.1                                                   Sterol Esterrs, Sterols and Unsaturated Fatty Acid                             Cholesteryl Palmitate/25                                                                     Triolein OR    0/0                                                             Trilinolein/0.25                                                 Cholesteryl Stearate/25                                                                      Triolein OR    0/0                                                             Trilinolein/0.25                                                 Cholesteryl Oleate/25                                                                        Triolein/0.25  2.1/67                                            Cholesteryl Linoleate/25                                                                     Triolein/0.25  0.92/60                                           Cholesterol/12.5                                                                             Trilinolein/0.25                                                                              0.30/22                                           Cholesteryl Palmitate/12.5                                                     Cholesterol/20                                                                               Trilinolein/0.25                                                                              1.9/73                                            Cholesteryl Stearate/5                                                         Cholesterol/12.5                                                                             Trilinolein/0.25                                                                              0.52/69                                           Cholesterol Stearate/12.5                                                      __________________________________________________________________________      .sup.1 Added to basal medium of (g/L deionized water): 1.5 Ardamine pH,        0.5 Glucose, 0.05 Lecitin, 0.025 Cholesterol, 5 mM CA.sup.2+.                  .sup.2 Oospore yield per 108 ml of medium.                               

The following Table 2 illustrates the effect of cations on in vitro oosporogenesis of L. giganteum.

                  TABLE 2                                                          ______________________________________                                         EFFECT OF CATIONS ON OOSPOROGENESIS                                                                  Oospore Yield                                                        Concentra-                                                                               (× 10.sup.-6).sup.2 /                              Added Cation                                                                               tion (mM) % Viability                                              ______________________________________                                         None, 2 mM EGTA.sup.3                                                                       0          0/0                                                    Ca.sup.2+    5        4.6/68                                                   Ca.sup.2+   10        2.1/85                                                   Ca.sup.2+   20        1.3/86                                                   Ca.sup.2+   30        5.6/17                                                   Mg.sup.2+    1        2.2/52                                                   Mg.sup.2+    5        4.9/53                                                   Mg.sup.2+   10        4.1/67                                                   Mg.sup.2+   20        4.4/81                                                   Mg.sup.2+   30        3.6/83                                                   Co.sup.2+, Mn.sup.2+, Ba.sup.2+,                                                           1-10      TOXIC OR NO                                              K.sup.+, Na.sup.+     OOSPORES PRODUCED                                        ______________________________________                                          .sup.1 Added as chloride salts, with variable degrees of hydration to          basal growth medium of (G/L) deionized water: 1.5 Ardamine pH; 1.5             Glucose; 0.05 Lecithin; 0.025 Cholesterol; 0.5 Trilinolein.                    .sup.2 Oospore yield per 108 ml of medium.                                     .sup.3 EGTA (ethyleneglycolbis-((3-amino-ethyl ether)N,N--tetraacetic          acid): a calcium chelator which binds the calcium ions present in low          concentrations in the growth media.                                      

Quantitative oosporogenesis tests were conducted utilizing various sterols in the growth medium. Hyphal segment transfers onto PYGLC (see Table 3 for media) were made and zoospore induction initiated between 7 and 12 days after the transfer. Each 10 cm diameter petri dish, containing 25 ml of the PYGLC medium, was inoculated with between 4×10⁴ and 6×10⁴ zoospores. Four replicates using each type of inoculation medium shown in Table 3 were performed. For different series of runs under the prescribed conditions, the mean oospore yield for 4 plates of a given medium usually varied by less than 10%. Cultures of L. giganteum used in the quantitative experiments shown in Table 3 had been maintained in vitro for less than 3 months. Quantitative oospore estimates were obtained after a minimum of two weeks growth on a given medium by adding distilled water to a beaker containing the agar pad from one petri dish to obtain a total volume of 100 ml, homogenizing the pad at high speed using an Osterizer blender. Homogenization was followed by oospore counting using the improved Neubauer hemacytometer.

                  TABLE 3                                                          ______________________________________                                         Effect of Sterol Structure on in vitro                                         Oosporogenesis by L. giganteum                                                 Inoculation Medium                                                                         No. of        No. of Viable                                        Sterol Supplement.sup.a                                                                    Oospores (× 10.sup.-5).sup.b                                                           Oospores (× 10.sup.-5).sup.c                   ______________________________________                                         cholesterol  2.8 ± 0.62                                                                                1.6 ± 0.40                                       7-dehydrocholes-                                                                            1.9 ± 0.55                                                                                1.0 ± 0.38                                       terol                                                                          sitosterol   2.8 ± 0.56                                                                               0.42 ± 0.26                                       desmosterol  2.6 ± 0.65                                                                                1.1 ± 0.31                                       fucosterol  0.42 ± 0.07                                                                               0.17 ± 0.19                                       stigmasterol                                                                               0.38 ± 0.10                                                                               0.16 ± 0.12                                       stigmastadienone                                                                           0.12 ± 0.10                                                                               0                                                    campesterol *             0                                                    erogesterol *             0                                                    lanosterol  *             0                                                    cholesteryl  2.7 ± 0.14                                                                               0.58 ± 0.29                                       acetate                                                                        cholesteryl 0.25 ± 0.14                                                                               0.09 ± 0.12                                       palmitate                                                                      20-hydroxyecdysone                                                                         0             0                                                    ______________________________________                                          .sup.a Basal growth medium, PYG, was supplemented with 50 ppm lecithin an      25 ppm of each sterol. PγG: 1.25 g/l Difco BactoPeptone, 1.25 g/l        Difco Yeast Extract, 3.0 g/l glucose, 0.1 g/l CaCl.sub.2.2H.sub.2 O, 15        g/l Difco agar. PYGLC: PYG plu s 0.05 g/l lecithin, 0.05 g/l cholesterol.      .sup.b Mean ± std. dev. per 10 cm diam. petri dish.                         *Oospores present, but less than 10.sup.2 per 10 cm diam. petri dish.          .sup.c Mean ± std. dev. per 10 cm diam. petri dish.                   

As indicated in Table 3 cholesterol was the most effective sterol supplement for producing viable oospores. Other cholesterol-like sterols, 7-dehydrocholesterol, sitosterol, desmosterol, fucosterol, stigmasterol, cholesteryl acetate and cholesteryl palmitate also produced viable oospores.

As may be seen from Table 1, the presence of certain fatty acids and fatty acid derivatives resulted in viable oospores. The enhancement of the production of viable L. giganteum oospores by these certain fatty acids and fatty acid derivatives is believed to be unique and unexpected among oomycetous fungi. 

Having described the present invention and various embodiments thereof, we claim:
 1. A method for in vitro induction of oospores comprising the step of: incubating zoospores or hyphal segments of Lagenidium giganteum in a culture medium under effective, oospore-inducing, incubation conditions comprising an assimilable source of carbon and an assimilable source of nitrogen; an oospore-inducing, effective amount of a sterol selected from a group consisting of cholesterol, 7-dehydrocholesterol, sitosterol, desmosterol, fucosterol, stigmasterol, cholesteryl acetate and cholesteryl palmitate; an oospore-inducing, effective amount of a non-toxic salt selected from the group consisting of calcium salts, magnesium salts, and mixtures thereof; and an oospore-inducing, effective amount of a compound selected from the group group consisting of oleic acid, palmitoleic acid, oleic acid esters, palmitoleic acid esters, linoleic acid esters, diolein, triolein, trilinolein and mixtures thereof.
 2. A method according to claim 1 wherein said sterol is cholesterol.
 3. A method according to claim 2 wherein said non-toxic salt is selected from the group consisting of calcium chloride and magnesium chloride.
 4. A method according to claim 3 wherein said culture medium comprises calcium chloride, glucose, lecithin, pH buffer and a compound selected from the group consisting of palmitoleic acid, diolein, triolein, trilinolein and mixtures thereof. 